Field of the Invention
The present invention describes the formation of stable, non-toxic nanoparticles that incorporate small interfering ribonucleic acids (siRNAs) and a method of using the nanoparticles for systemic treatment of disease.
Background
Many diseases are caused by genetic mutations that lead to abnormal cell function or can be treated by reducing the expression of a targeted gene. In particular, it is believed that a variety of diseases such as genetic, infectious, cardiovascular, metabolic and immune-mediated diseases as well as diabetes, osteoporosis and cancer may be more effectively treated by reducing the expression of specific gene products.
For example, the vast majority of pharmaceutical drugs used to treat cancers utilize unencapsulated free compounds to alter cell function or kill abnormal cells. Virtually all of these treatments utilize non-specific mechanisms of action and have poor biodistribution profiles that result in toxic side effects to healthy tissues. This often occurs from drugs interacting with any cell with which they come in contact, causing significant cell death and toxicity in non-diseased cells. In fact, severe side effects from drugs often limit the dosage that patients can receive. For some diseases, including cancers, this leaves severely ill patients with little choice but to endure widespread toxicity for small gains in therapeutic efficacy.
The elucidation of specific signaling mechanisms and cross-talk between pathways has led to the advent of targeted therapies. These targeted therapies, which interfere with specific molecular pathways, include monoclonal antibodies and small molecule therapeutics. While targeted therapies have improved treatment and survival outcomes, toxic side effects resulting from the inhibition of normal cell function still exist. In addition, infectious organisms and cancerous cells have the ability to “escape” or acquire resistance to therapies by accumulating mutations and altering metabolism during disease progression.
The ability to treat the direct cause of disease, disease-causing genetic mutations, overexpression or infectious agents would provide a therapeutic modality that is only active in the desired cells, providing further specificity that could greatly decrease side effects.
Currently genetic-based therapeutic approaches, including ribonucleic acid interference (RNAi) mediated by small interfering ribonucleic acid (siRNA), are limited by systemic degradation, poor biodistribution and limited cellular uptake. Particularly for systemic administration, the ability to concentrate siRNAs to achieve therapeutic threshold concentrations in the desired tissues remains difficult. Non-toxic nanoscale delivery modalities currently offer the best chance to achieve therapeutic dose levels of siRNA. This is particularly true for cancer applications due to localization of nanoparticles and their associated payloads to solid tumors via the enhanced permeability and retention effect (EPR)1-3.
Related Art
The design and engineering of siRNA delivery systems has recently been pursued by several groups. Tekmira and Alnylam utilize Solid Nucleic Acid Lipid Nanoparticle (SNALP) technology, which utilizes cationic or charge-conversional lipids with polyethylene glycol (PEG) surface groups, and are currently in early clinical trials4-10. The biodistribution of this delivery system mainly targets the liver, limiting the cancer applications and also causing liver toxicity11.
Calando has developed a cyclodextrin-based delivery system which has proven immunogenic in early clinical trials and difficult to manufacture12-15.
Silence Therapeutics AtuPlex lipid-based delivery system is currently undergoing early clinical studies16-19.
Several academic groups have explored the use of calcium phosphate nanoparticles for siRNA delivery20-31. However, these methods do not teach the synthesis of calcium phosphate-siRNA nanoparticles that do not include other, potentially toxic components, such as residual buffers (Tris & HEPES) or synthesis components (surfactants). From a pharmaceutical standpoint, toxicity can be a limiting factor to drug development and eliminating potential toxicity through the exclusion of unnecessary components has been a goal not previously achieved in designing a drug delivery system.
Each of the prior art approaches to produce calcium phosphate nanoparticles for siRNA delivery has run into a problem. For example, Epple et al teaches the use of siRNA as the dispersant. It has been noted that doing so will likely compromise dispersion in vivo28. Huang et al, utilizes a difficult to purify microemulsion system as well as cationic lipids for dispersion20,21,24,25,30,31, which will limit biodistribution. The microemulsion synthesis also involves materials known to be toxic that may be incorporated into the resulting nanoparticles. Kataoka et al, employs exotic and potentially toxic charge conversional block co-polymers26,27 for dispersion. In addition, Kataoka's particles are precipitated in the presence of buffers such as HEPES and Tris22,23,26-29. Tris is known to be a toxic compound, therefore the association of these molecules with the calcium phosphate nanoparticles can be expected to cause toxicity. None of the above referenced groups has shown a detailed molecular analysis of their particles to demonstrate the lack of incorporation of residual, toxic components.
Another approach was explored by a group at the University of Tokyo that used double stranded siRNA conjugated to PEG via a disulfide bond to provide dispersion29. As shown in that work29, as well as work done by the present inventors, the particle architecture resulting from Zhang's method is colloidally unstable, particularly in the presence of serum, and therefore not suitable for therapeutic development.